The long-term objectives of this project are to understand the structure- function relationship of the Dbl family guanine nucleotide exchange factors (GEFs) and to determine the mechanism of signal transduction involving the Dbl family proteins. The Dbl related GEFs represent a fast growing family of cell growth regulatory molecules with over 50 mammalian members. Their cellular functions intimately depend on their ability to interact and activate specific Rho GTPases leading to various physiological responses, including cytokinesis, cell movement, cell proliferation and differentiation, and apoptosis. All Dbl family members share the structural array of a central Dbl-homology (DH) domain in tandem at the carboxyl terminus with a Pleckstrin-homology (PH) domain. Previous studies have established that DH domain in these proteins is responsible for the Rho GTPase binding and the GEF activities, while the PH domain, together with additional structural motifs, is involved in intracellular targeting and/or modulation of the DH domain function. To continue the lines of studies of the last funding period, we plan to pursue three specific aims to further dissect the structural features of Dbl family GEFs and to delineate their regulatory and signaling mechanisms in this proposal. In the first aim we will identify the sites of DH domain involved in specifying substrate interaction and generate dominant negative GEFs to be used in delineation of their downstream pathways. In the second aim we will examine the intra- and inter-molecular interactions of Dbl family members involving the DH, PH, and additional regulatory domains, focusing on the regulatory role such interactions may bear on the GEF function. In the third aim we will examine the activation mechanism of Dbl-family GEFs in response to agonist stimulation by investigating the relationship between GEF translocation and activation and by determining the involvement of heterotrimeric G-proteins, protein kinases, or PI3-kinase in the process. These biochemical studies will provide valuable information on how the Dbl family growth regulators contribute to Rho GTPase activation, which may underline the principles of a variety of human pathological conditions including cancer, inflammation, and developmental disorder.